Uptake and Bioaccumulation of Endocrine Disruptors and Pharmaceutical compounds … · 2019-02-07 · Final extracts analyzed by UPLC/QTRAP 5500 MS 0.5 g of freeze-dried fish & mussel

Uptake and Bioaccumulation of Endocrine Disruptors and

Pharmaceutical compounds in Biofilm, Macroinvertebrates, and Fish in four Mediterranean Rivers

Belinda Huerta, Victoria Osorio, Marina Gorga, Anna Jakimska, Nuria de Castro, Lidia Ponsati, Isabel Muñoz, Sandra Pérez, Mira Petrovic, Sara Rodríguez-Mozaz, Damià Barceló

INTRODUCTIONINTRODUCTION

OBJECTIVESOBJECTIVES

ANALYTICAL METHODOLOGIESANALYTICAL METHODOLOGIES

PRESENCE OF PRESENCE OF PhACsPhACs & & EDCsEDCs IN BIOTAIN BIOTA

CONCLUSIONSCONCLUSIONS

INTRODUCTIONINTRODUCTION

Introduction

WHO-State of the Science of Endocrine Disrupting Chemicals - 2012

Introduction

WHO-State of the Science of Endocrine Disrupting Chemicals - 2012

Disruption of endocrine system

Biologically active

Highly potent at low levelsUNPREDICTED

ENVIRONMENTAL IMPLICATIONS• acute & chronic toxicity

• antibiotic resistance

• genotoxicity

• endocrine disruption

At environmental concentrationshas been seen that…

• synthetic hormone 17α-ethinylestradiol (EE2) has induction of estrogenic effects in fish

• diclofenac led to tissue damage in fish

• behavioural alterations in fish after exposure to sertraline

Introduction

PhACs

EDCs

Few studies related to uptake of pharmaceuticals in freshwater organisms

Organisms such as invertebrates or biofilm possess the capacity to integrate environmental variations. They are also at the very bottom of the trophic chain.

Fish considered to be a feasible organisms for pollution monitoring, astheir biological particularities make them potentially susceptible to pharmaceutical bioaccumulation (ubiquitous, role as carrier of energy, lifespan)

Introduction

PREVIOUS FIELD STUDIESPREVIOUS FIELD STUDIES

Introduction

Pharmaceutical Active Compounds PhACs

Endocrine Disrupting Compounds EDCs

Brooks et al. 2005Fluoxetine andsertraline in fish braintissue (max 30ng/g)

Ramirez et al. 2007Diphenydramine, diltiazemcarbamazepineIn fish

Li et al. 2012Antibiotics in Mollusks

Ramashwamy et al. 2011TBEP & Triclosan in fish bile

Rüdel et al. 2013Triclosan in fish

Coogan et al. 2007Triclosan in algae

Brodin et al. 2013Oxazepam in fishChu & Metcalfe

2007FluoxetineIn fish

OBJECTIVESOBJECTIVES

To develope an analytical methodology for the analysis of PhACs & EDCs in:

- fish

- macroinvertebrates

- biofilm

To characterize the presence and bioaccumulation of PhACs and EDCs in aquatic organisms from different trophic levels – biofilm, two macroinvertebrates: Hydropsyche sp, (collector-filterer insect larvae) and Dreissena polymorpha (zebra mussel), and fish – collected from four rivers basins in Spain: Ebro, Llobregat, Júcar and Guadalquivir.

Objectives

ANALYTICAL METHODOLOGIESANALYTICAL METHODOLOGIES

PLE Extraction

GPC Clean-up

Analysis by UPLC-MS/MS

PhACsPhACs EDCsEDCs

QuEChERSExtraction & Purification


1 g of freeze-dried fish or 0.5 g of mussel extracted using PLE

Extracts purified in a GPC system for lipid removal

Final extracts analyzed by UPLC/QTRAP 5500 MS

0.5 g of freeze-dried fish & mussel extracted in ACN/H2O) after addition of extraction salts

Extracts purified using dSPE


Analytical Methodology: Fish

Huerta et al. J of Chrom. (2013) Jakimska et al. J of Chrom. (2013)

PhACsPhACs & & EDCsEDCsExtraction by sonication

Purification by Ostro 96-well plate

100 mg of freeze-dried sample placed in a 50-mL Falcon tube + 3 ml of MeOH

Sonication applied in 3 cycles of 120 s, with amplitude (intensity) of 30 %

Samples centrifuged at 11000 rpm, for 5 min and supernatant collected

Extracts redissolved in H2O/ACN (1:3, 1 % HCOOH, v/v)

Extracts added to an OstroTM 96-well plate

After extraction by vacuum, purified extracts were collected



Analytical Methodology: Macroinvertebrates

PhACsPhACs & & EDCsEDCsPLE Extraction


SPE Purification

200 mg of freeze-dried sample placed in 10 ml ASE 350 cells

Extraction applied in 3 cycles of 5 min, with a mixture of citric buffer (pH 4)/ACN (1:1) as solvent and a Tª=60 ºC

Extracts were diluted to 100 ml and purified by SPE Oasis HLB (6ml, 200 mg)

Eluates in MeOH collected and evaporated


Analytical Methodology: Biofilm

PLE Extraction

GPC Clean-up


Fish:PhACsFish:PhACs Fish:EDCsFish:EDCs MacrMacr: : EDCsEDCs & & PhACsPhACs

Biofilm:EDCsBiofilm:EDCs& & PhACsPhACs


QuEChERS Extraction& Purification

Extraction by sonication

Purification by Ostro 96-well plate


PLE Extraction


SPE Purification

• Nº compounds: 20 PhACs

• Main challenges:

- lipid content

- trace levels

• Nº compounds: 19 EDCs


- lipid content

- trace levels

• Nº compounds: 41 PhACs + 21 EDCs


- sample size

- variety

• Nº compounds: 43 PhACs + 14 EDCs


- sample size

Analytical Methodology: Summary

PRESENCE OF PHACS & EDCS IN PRESENCE OF PHACS & EDCS IN BIOTABIOTA

Sampling Campaign

8 compounds detectedFamilies: Psychiatric drugs, non-steroidal anti-inflammatory drugs (NSAID) and β-blockersDiclofenac found in 4 riversMost polluted river basin: LlobregatHighest conc. diclofenac in LLO6, after theentrance of Anoia (highly contaminanted by industrial WW)Ebro: no clear pollution gradient in the 5 sites PhACs detected in 4 sitesJucar & Guadalquivir: ↓ PhACs conc. Onlydetected in fish in 1 site (GUA4 and JUC5) of each riverNo difference among species, according totheir feeding habits, age or niche

11 compounds detected

Families: Parabens, triazoles, flameretardants, antibacterial, plasticizer

The most ubiquitous contaminants in fish samples was the flame retardant TBEP, found inthe 75% of all samples analyzed at values up to 52.96 ng/g.

Llobregat higher levels of EDCs (in river and fish homogenates) TBEP levels and extremely high levels of BPA (223.91 ± 11.51) in in LLO5

Jucar and Guadalquivir river samples were comparatively less polluted, except by thesampling point GUA4, where high level of BPA (59.09 ± 8.12) was determined

PhACsPhACs EDCsEDCs

Results: Fish

PhACsPhACs EDCsEDCs

Two species: Trichoptera & Zebra mussels (only in Ebro)

4 compounds detected: 4 (citalopram, carbamazepine, diclofenac, levamisole) in mussel and 1 (diclofenac) in trichoptera

Families: Psychiatric drugs, non-steroidal anti-inflammatory drugs (NSAID) and anthelminthic

Conc. in mussel < 3 ng/g

Conc. in trichoptera higher than fish (50 ng/g)

Diclofenac was found in samples fromall the river basins

Two species: Trichoptera & Zebra mussels (only in Ebro)

4 compounds detected: 2 in mussel(TBEP, methylparaben) and 3 in trichoptera (TBEP, benzylparaben, nonylphenol)

Families: Parabens, alkylphenols and flame retardants

Conc. parabens < 3 ng/g

Mean conc. of TBEP and nonylphenol50-70 ng/g (d.w.)

Detection frequency of TBEP: 100%

Results: Macroinvertebrates

PhACsPhACs EDCsEDCs

Samples from Ebro & Llobregat only

No compounds in Ebro

15 compounds (4 metabolites) detectedin Llobregat

Clear pollution gradient: 0 compoundsdetected in LLO3 to 15 compounds in LLO7

Families: Psychiatric drugs, non-steroidal anti-inflammatory drugs (NSAID), antibiotics, diuretics, lipid regulators and β-blockers

Max. conc. for sertraline (230 ng/g, dw)

Samples from Ebro & Llobregat only

5 compounds detected

Detection frequency: 90 – 100%

Families: Parabens, flame retardants, plasticizer

Similar levels in both river basins

Conc. range between nd – 270 ng/g (dw) (case of Bisphenol A in Ebro)

Average conc. 50 ng/g (dw)

Results: Biofilm

RESULTS: SUMMARYRESULTS: SUMMARY

PhACsPhACs

EBRO

• No clear pollution gradient in the 5 sites

• Non-detected in biofilm

• 4 compounds in macroinv. & fish

GUADALQUIVIR

• Lower levels in water &sediment↓ biota

• No data for biofilm

• Occurrence in GUA4 after WWTP in fish and macroinv.

JUCAR



• Occurrence in JUC5 in fish (no data for macroinv. In JUC5 & JUC6)

LLOBREGAT

• Evident pollution gradient fromLLO3 to LLO7

• Biofilm as the species thataccumulates > nº compounds (15)

• Fish & Macroinv: 3 compounds

• Metabolites of carbamazepineand β-blockers detected in biofilm

• Psychiatric drugs, non-steroidal anti-inflammatory drugs as the most common families

• Concentration: Biofilm> Macroinv. > fish

RESULTS: SUMMARYRESULTS: SUMMARY

EDCsEDCs

EBRO

• No clear pollution gradientin the 5 sites

•↑↑ BPA in biofilm

• TBEP detection frequency in all the organisms: 100%

• Highest nº compounds detected in fish (6)

GUADALQUIVIR



• GUA4: ↑ BPA in fish

• TBEP detected in 100% of samplesin fish and biofilm

JUCAR



• TBEP detected 95-100 % of fishand macroinv. samples

LLOBREGAT

• Higher levels of EDCs

• TBEP detection frequency in all the organisms: 75-100%

• ↑↑ BPA in fish and biofilm

• Parabens detected in 100 % of biofilm and fish samples

• Parabens, flame retardants, and plasticizer as the most common families

• Concentration: Biofilm ~ Macroinv. ~ fish


Methodologies for the analysis of PhACs (20-43) & EDCs (17-21) weredeveloped for 3 aquatic organisms: fish, macroinvertebrates and biofilm.

A total of 80 biota samples were analyzed in the four rivers

Up to 15 PhACs and 11 EDCs were detected in biota

Psychiatric drugs, non-steroidal anti-inflammatory drugs for PhACs and parabens, flame retardants, and plasticizer for EDCs as the most common families

Highest concentrations of PhACs found in biofilm, while for EDCs differences among species are not so obvious

Pollution gradient: Llobregat > Ebro > Jucar > Guadalquivir

NEXT STEPS:

Establish relations between concentration in biota with that in water and sediment

Investigate the presence of metabolites and transformation products of target pollutants in the samples


Thank you all for your attention

Assessing and predicting effects on water quantity and quality in Iberian rivers caused by global change (SCARCE)Consolider‐Ingenio 2010 CSD2009‐00065 SCARCE Spanish Ministry of Economy and Competitiveness

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Uptake and Bioaccumulation of Endocrine Disruptors and Pharmaceutical compounds … · 2019-02-07 · Final extracts analyzed by UPLC/QTRAP 5500 MS 0.5 g of freeze-dried fish & mussel